GCN4 is a transcriptional activator of amino acid biosynthetic genes in yeast that is regulated at the translational level by the eIF2alpha kinase GCN2. The translational efficiency of GCN4 mRNA is coupled to the binding of initiator methionyl tRNA to the ribosome, so that GCN4 expression is a sensitive functional indicator of translation factors involved in this step of translation, including eIF2, its guanine nucleotide exchange factor eIF2B, eIF5, and components of eIF3. Phosphorylation of eIF2 by GCN2 under amino acid starvation conditions stimulates GCN4 translation by inhibiting guanine nucleotide exchange on eIF2 by eIF2B, thereby reducing the concentration of the ternary complex eIF2/GTP/Met-tRNAiMet. The GCN3, GCD7, and GCD2 subunits of eIF2B comprise a regulatory subdomain that mediates inhibition of eIF2B by phosphoryated eIF2. Mutations in homologous segments of these subunits abolish the inhibitory effect of eIF2 phosphorylation on nucleotide exchange by eIF2B. Biochemical analysis has shown that these mutations allow phosphoryated eIF2 to be accepted as substrate without substantially altering the affinity of eIF2B for eIF2. The GCN3/GCD7/GCD2 subcomplex can interact with purified eIF2 and shows greater affinity for the phosphorylated form of the protein. The GCD1 and GCD6 subunits of eIF2B can also form a stable subcomplex that binds with equal affinity to phosphorylated and nonphosphorylated eIF2. Thus, eIF2B contains two independent binding sites for eIF2, only one of which is sensitive to eIF2 phosphorylation. Purification of eIF3 using an affinity-tagged form of the PRT1 subunit, coupled with mass spectrometric analysis of the copurifying proteins, revealed a core complex of 5 subunits that is conserved between yeast and human eIF3. eIF5 was found stably associated with yeast eIF3, suggesting that recruitment of eIF5 to the ribosome is mediated by eIF3. GCD10 and GCD14 are components of a nuclear complex required specifically for the processing and stability of initiator tRNAMet, suggesting a novel mechanism for regulating translation initiation. The activation of GCN2 kinase function by uncharged tRNA appears to involve dimerization, multiple contacts between the kinase domain and flanking regulatory domains in GCN2, and autophosphorylation of the activation loop in the kinase domain. Inhibition of the double-stranded RNA-dependent human eIF2alpha kinase PKR by vaccinia virus E3 protein was reconstituted in yeast and shown to be dependent on the N-terminal domain of E3 in addition to its double-stranded RNA binding domain. The transcriptional activation domain in GCN4 contains hydrophobic clusters that mediate physical interactions with subunits of RNA Polymerase II holoenzyme, TFIID and the ADA/GCN5 coactivator. It was shown that adenine repression of ADE5 transcription occurs by preventing a physical interaction between the BAS1 and BAS2/PHO2 proteins which unmasks a latent activation function in BAS1.